CN113528461B

CN113528461B - Isolated aeromonas salmonicida phage, compositions and uses thereof

Info

Publication number: CN113528461B
Application number: CN202110802404.2A
Authority: CN
Inventors: 刘琴; 张元兴; 王启要; 徐蓁禾; 刘晓红; 邵帅; 王蓬勃
Original assignee: Shanghai Weizhong Biotechnology Co ltd; East China University of Science and Technology
Current assignee: Shanghai Weizhong Biotechnology Co ltd; East China University of Science and Technology
Priority date: 2021-07-15
Filing date: 2021-07-15
Publication date: 2023-10-24
Anticipated expiration: 2041-07-15
Also published as: CN113528461A

Abstract

The application provides an isolated aeromonas salmonicida phage, a composition and application thereof. The present inventors have screened for a phage that can kill aeromonas salmonicida with high efficiency, has strong infectivity and strong lytic efficacy, and has a short incubation period in bacterial hosts. The phage of the present application can be applied in agriculture, medicine, environment, etc. to combat its bacterial host, which has a good development prospect as an antibiotic substitute.

Description

Isolated aeromonas salmonicida phage, compositions and uses thereof

Technical Field

The application belongs to the fields of biotechnology and microbiology, and more particularly relates to an isolated aeromonas salmonicida phage, a composition thereof and application thereof.

Background

In recent years, with rapid development of the aquaculture industry, the aquaculture scale is expanding. With the continuous popularization of intensive and large-scale cultivation modes, the method is more and more important for preventing and treating diseases of aquatic animals, and effective prevention and treatment can promote the development of cultivation industry. Bacterial diseases are taken as important aquatic diseases, and the effective control of the diseases can promote the orderly operation of the cultivation industry and avoid the economic loss caused by large-scale diseases.

Aeromonas salmonicida (Aeromonas salmonicida) is a gram-negative short bacillus, belongs to aeromonas psychrophilia, and is widely distributed in aquatic environments. Aeromonas Salmonicida includes five subspecies such as achromonas (achromogenies), salmonicida subspecies (Salmoniida), japanese Salmonicida subspecies (Masoucida), smith subspecies (Smithia), and pectogenous subspecies (Pectinolytica). The bacteria is a main pathogenic bacteria causing fish scabies or ulcer, and can be transmitted via skin, gill, mouth and blood to infect various fishes such as salmonidae, cypriidae, finidae, anguilidae, paralichthys, and Pteriidae. At present, aeromonas salmonicida is controlled mainly by means of antibiotics in aquaculture. However, the large-scale use of antibiotics is accompanied by the appearance of multi-drug resistant bacteria, which affects the quality safety of aquatic products.

Thus, there is a need in the art for antibiotic alternatives for disease control of multi-resistant aeromonas salmonicida to reduce antibiotic usage.

Disclosure of Invention

The application aims at providing an isolated aeromonas salmonicida phage, a composition and application thereof.

In a first aspect of the application, there is provided an isolated phage having a collection number of CCTCC NO: M2021631 at the China center for type culture collection.

In a preferred embodiment, the phage has a characteristic comprising a member selected from the group consisting of:

(a) The head part is an icosahedron;

(b) Belonging to the family of myeocaudaceae of the order of the end phageales;

(c) The pH-resistant water-based paint has adaptability to acidic and alkaline environments and tolerates the environment with the pH of 3-11; preferably in the pH range of 4.0 to 9.0, more preferably in the pH range of 5.0 to 8.0;

(d) Has adaptability to high temperature and low temperature, tolerance is 0-50 ℃; preferably in the range of 0 to 40 ℃, more preferably in the range of 0 to 30 ℃ (e.g. 5, 10, 15, 20, 25, 28 ℃);

(e) The plaque is transparent and round, the periphery is provided with halation, and the diameter is about 3.0-3.5 mm;

(f) The incubation period in the host is 10-30 minutes; preferably 15 to 25 minutes; more preferably 18 to 22 minutes;

(g) The infection complex number is 100:1-1:1000; preferably 1:1 to 1:100; more preferably 1:10 (0.1);

(h) The cracking amount for aeromonas salmonicida is 100-500 PFU/cell; preferably 200 to 400 PFU/cell (e.g., 250 to 300 PFU/cell);

(i) The lysis rate against aeromonas salmonicida is over 95%; preferably more than 97%;

(j) The titer of the culture is higher than 10 ⁸ PFU/mL; preferably higher than 5 x 10 ⁸ PFU/mL。

In another preferred embodiment, the phage described in (a) has a head length of 95.+ -.10 nm, a width of 91.+ -.10 nm and a tail length of 109.+ -.10 nm.

In another preferred embodiment, the phage has a head length of 95.+ -.5 nm, a width of 91.+ -.5 nm and a tail length of 109.+ -.5 nm.

In another preferred embodiment, in (c), an environment of pH3-10 is tolerated.

In another aspect of the application, there is provided the use of said phage for inhibiting its bacterial host; or for the preparation of a composition for inhibiting a bacterial host thereof; wherein the bacterial host is aeromonas salmonicida.

In a preferred embodiment, the application is a non-therapeutic application; for example against a host of said phage in the environment, such as aeromonas salmonicida.

In another preferred embodiment, the composition is a pharmaceutical composition, a food composition, a feed composition, a detergent and/or a disinfectant.

In another preferred embodiment, the pharmaceutical composition is a vaccine composition; more preferably, the vaccine composition prevents fish diseases associated with bacterial host infection of said phage.

In another preferred embodiment, the pharmaceutical composition is a therapeutic composition; more preferably, the therapeutic composition treats a fish disease associated with bacterial host infection by said bacteriophage; more preferably, the fish disease comprises: scabies caused by aeromonas salmonicida infection.

In another preferred embodiment, in the feed composition, the phage is used as a feed additive.

In another preferred embodiment, the cleaning or sanitizing agent is an environmental cleaning or sanitizing agent.

In another preferred embodiment, the fish is marine fish.

In another aspect of the application, there is provided a composition for inhibiting bacteria comprising an isolated phage as described in any of the preceding.

In a preferred embodiment, it further comprises a biologically acceptable carrier.

In another preferred embodiment, the phage in the composition is greater than or equal to 10 ³ PFU/mL。

In another preferred embodiment, the phage in the composition is 10 ³ ～10 ¹⁰ PFU/mL。

In another preferred embodiment, the phage in the composition is 10 ⁴ ～10 ⁹ PFU/mL, e.g. 10 ⁵ 、10 ⁶ 、10 ⁷ 、10 ⁸ PFU/mL。

In another aspect of the application, there is provided a method of preparing a composition of phage as described in any of the preceding, comprising: amplifying said phage; mixing with a carrier, wherein the carrier is a biologically acceptable carrier.

In a preferred embodiment, the carrier includes, but is not limited to: solvents, adjuvants, buffers, lyoprotectants, wetting agents, osmotic agents, dispersing agents, emulsifiers, stabilizers, adhesion agents, bulking agents, adjuvants, surfactants or controlled release agents.

In another preferred embodiment, the amplification culture comprises: inoculating the phage to its bacterial host culture (broth) so that the phage infects its host for replication; preferably, the bacterial host is aeromonas salmonicida.

In another preferred embodiment, the pharmaceutical composition is a vaccine composition.

In another preferred embodiment, the vaccine composition prevents fish diseases associated with bacterial host infection by said phage.

In another preferred embodiment, the pharmaceutical composition is a therapeutic composition.

In another preferred embodiment, the therapeutic composition treats a fish disease associated with bacterial host infection by said bacteriophage.

In another preferred embodiment, the fish disease comprises: scabies caused by aeromonas salmonicida infection.

In another preferred embodiment, the phage formulation comprises a dosage form including (but not limited to): liquid or solid formulations; more preferably comprises: freeze-dried agent, water solution, emulsion, sprayable solution, oily or aqueous dispersion, suspending agent, powder, granule, wettable powder, emulsifiable concentrate or microgel.

In another preferred embodiment, the composition is at a pH of 3.0 to 11.0, more preferably at a pH of 4.0 to 9.0 (e.g., at a pH of 5.0 to 9.0, more particularly at a pH of 6.0, 6.5, 7.0, 7.5, 8.0, 8.5).

In another aspect of the application, there is provided a method of inhibiting a pathogenic microorganism or of killing an insect, the method comprising: treating a subject in need of bacterial inhibition with a phage of any of the foregoing; the bacteria are bacteria which can be infected (infested) by the phage and then lysed; preferably, the bacterium is aeromonas salmonicida.

In a preferred embodiment, the treatment is carried out at a pH of 3.0 to 11.0, more preferably at a pH of 4.0 to 9.0 (e.g., pH5.0 to 9.0, more particularly at a pH of 6.0, 6.5, 7.0, 7.5, 8.0, 8.5);

in another preferred embodiment, the treatment is carried out at a temperature of from 0 to 50 ℃, more preferably from 0 to 40 ℃, still more preferably from 0 to 30 ℃ (e.g. 5, 10, 15, 20, 25, 28 ℃).

In another preferred embodiment, the uses and methods are non-therapeutic uses and methods that do not involve direct prophylaxis or treatment of a subject (e.g., decontamination of a pathogen-containing area (e.g., public, fish farming water)/appliance, etc., or decontamination of food, feed, etc., that may be attached by a pathogen).

In another aspect of the application there is provided a kit/kit for inhibiting bacteria, the kit/kit comprising: phage of any of the preceding; or a composition as described in any of the preceding.

Other aspects of the application will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

The above and other features, properties and advantages of the present application will become more apparent from the following description of the embodiments in conjunction with the accompanying drawings:

FIG. 1, plaque map of phage vB_ AsM _ZHF of the application.

FIG. 2, electron microscopy images of phage vB_ AsM _ZHF of the application.

FIG. 3, a graph of the results of a complex experiment of optimal infection with phage vB_ AsM _ZHF according to the application.

FIG. 4, one-step growth profile of phage vB_ AsM _ZHF of the application.

FIG. 5, a graph of the results of the acid-base resistance experiment of phage vB_ AsM _ZHF of the present application.

FIG. 6, a graph of the results of the thermal stability experiments of the phage vB_ AsM _ZHF of the application.

FIG. 7 shows the results of the cleavage curve of the phage vB_ AsM _ZHF of the application against the host bacterium AS01.

FIG. 8 is a graph showing the experimental results of the therapeutic effect of phage vB_ AsM _ZHF of the present application on host bacterium AS01 on turbot.

Detailed Description

The inventor is dedicated to research on bacterial resistance and disease prevention of fishes, and in a large number of screening works, a strain of aeromonas salmonicida is obtained, and then the aeromonas salmonicida is separated and obtained to efficiently kill the aeromonas salmonicida. The phage of the application has strong infection capability, short incubation period in bacterial host and strong cracking effect. The phage of the present application can be applied in agriculture, medicine, environment, etc. to combat its bacterial host, which has a good development prospect as an antibiotic substitute.

Terminology

As used herein, the "pathogen" is a microorganism that is harmful to humans, animals, plants or the environment, especially a microorganism that is harmful to fish. More specifically, "pathogen" refers to a microorganism that is capable of being lysed/destroyed by a bacteriophage of the present application, including a bacterial host of a bacteriophage of the present application; preferably, the bacterial host is aeromonas salmonicida, and when the phage of the present application acts on the bacterial host, the host is lysed/destroyed.

In the present application, the term "comprising" means that the various ingredients can be applied together in the mixture or composition of the present application. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "containing.

As used herein, a "biologically acceptable carrier" is a carrier for delivering a bacteriophage of the present application to an object to be treated, including a pathogen host, the environment in which the pathogen is located (location, appliance, food, feed, etc.), or the environment in which the pathogen host is located. The carrier may be, but is not limited to: a pharmaceutically acceptable carrier, a feed acceptable carrier, and/or a chemically acceptable carrier. The carrier is generally an environmentally friendly or harmless solvent, suspending agent or excipient that is controllable in terms of toxicity, side effects, or is harmless to humans and animals. The carrier may be a liquid or a solid, and is preferably a carrier capable of maintaining the bioactivity of the phage of the present application to a high degree.

As used herein, "combination" means that more than one active substance is used in conjunction or combined together.

As used herein, when referring to a bacteriophage or a composition containing the same, the terms "lytic rate," "antimicrobial activity," and "antipathogenic activity," as used herein, are used interchangeably to refer to the ability to kill and/or inhibit the growth or proliferation of a pathogen (especially a bacterial host).

Phage and application thereof

Phage is a virus that uses bacteria as a host, and is widely available in nature and has a variety of types. The phage has the characteristics of high host specificity, low toxicity, capability of removing a biofilm, small damage to the micro-ecological environment and the like. After the phage infects the host bacteria, a large number of sub-generation phage can be generated and a new round of infection is carried out, so that the control effect can be achieved by using low-dose phage.

The inventor screens out a salmonicida aeromonas from diseased fishes in a turbot farm in a double-layer flat plate method, then separates out a salmonicida aeromonas phage from wastewater of the farm by taking the salmonicida aeromonas as a host, and names the phage obtained by natural separation as vB_ AsM _ZHF, and the preservation number is CCTCC NO: M2021631.

Further analysis by the present inventors showed that the phage included features selected from the group consisting of:

(a) The head part is an icosahedron;

(b) Belonging to the family of myeocaudaceae of the order of the end phageales;

The phage of the application is an active organism and once obtained, can be prepared in large quantities by means known in the art. This is usually released by contacting it with a bacterial host, by massive replication after invasion of the host cell, and finally by lysis of the host cell.

The phage of the application may be a naturally isolated vB_ AsM _ZHF, as well as variants thereof, e.g.by subjecting it to molecular genetic manipulation (e.g.genomic engineering) to obtain mutants thereof, thereby modulating/improving properties in a certain aspect thereof, e.g.by molecular genetic manipulation (altering the activity of one or more of its proteins) to further promote its ability to infect a host, to expand its host species, to shorten its latency in a host and/or to enhance its ability to lyse a host.

Namely, the phage vB_ AsM _ZHF can be used as a starting phage, and further improved by means of laboratory domestication, genetic breeding, molecular genetic operation and the like to obtain derivative phage with higher yield or higher activity. Further screening of the phage obtained by optimization by these manual means using vB_ AsM _ZHF of the present application as a starting phage is also included in the overall scope of the present application.

As an alternative, the application also provides an active biomolecule (e.g., an isolated phage polypeptide or active fragment, variant or derivative thereof) isolated from a phage of the application. Since the phage of the application is entirely new, its genome and the active polypeptide encoded by its genome can also be included in the application, potentially a polypeptide or combination of polypeptides thereof can also be used for use as a bacterial host for inhibition or other uses. The inhibition may be partial inhibition or total inhibition. For example, one, two or more polypeptides isolated from the phage can function as an invading cell; while the other one, two or polypeptide can exert a lytic effect.

The phage of the application has strong lytic effect on pathogenic bacterial hosts (such as aeromonas salmonicida) in aquaculture. According to the embodiment of the application, the cracking rate of the aeromonas salmonicida can reach 98%. Thus, phage sources can be provided for large-scale industrial production of phages, for inhibition of pathogenic bacteria (especially aeromonas salmonicida) in aquaculture.

According to the examples of the present application, the phage of the present application can maintain a good activity after incubation for 2 hours at pH ranging from 5.0 to 8.0, and at ph=11 and ph=3, the phage still has a certain activity, and the phage has good tolerance to both acidic environment and alkaline environment.

According to the examples of the present application, the phage of the present application remained at a titer of 10 after 2 hours of treatment at 50 ℃ ⁷ pfu/ml or more. It has good high temperature resistance and at the same time good stability at low temperatures.

The phage according to the application has a broad range of applications including, but not limited to, the following:

(1) Inhibit and inhibit the growth of a bacterial host (e.g., aeromonas salmonicida);

(2) Preparing a product that kills a bactericidal host (e.g., aeromonas salmonicida);

(3) Preparing a product that inhibits the growth of a bacterial host (e.g., aeromonas salmonicida);

(4) Preparing a product for preventing and/or treating fish diseases caused by a bacterial host (such as aeromonas salmonicida);

(5) Preparing a product for preventing and/or treating an inflammatory response caused by a bacterial host (e.g., aeromonas salmonicida);

(6) The bacteriophage of the application can be used as a feed additive or can be used for preparing an environment disinfectant as an effective sterilization component.

In the application, the fish is any fish that can be infected by the pathogen (bacterial host). For example, it may be a marine fish or a freshwater fish, preferably a marine fish.

The phage of the application may be used alone or in combination with one or more other types of phage.

When used, the phage or polypeptide of the application may be administered alone or in combination with a carrier, forming a composition.

Compositions/formulations/kits

The present application provides a composition comprising an effective amount of phage, and the balance a biologically acceptable carrier. The composition of the present application may additionally comprise excipients or stabilizers.

The dosage form of the composition may be a wide variety including, but not limited to: freeze-dried agent, water solution, emulsion, sprayable solution, oily or aqueous dispersion, suspending agent, powder, granule, wettable powder, emulsifiable concentrate or microcapsule.

It will be appreciated that dosage forms that are capable of delivering the phage of the application to a subject in need of treatment while retaining all or part of the activity are desirable. Those which are easy to deliver are preferred, and as some preferred means the composition may be a lyophilizate, liquid administration/injection, spray or aerosol.

The concentrated composition has a high content of active ingredient (polypeptide), such as 10 ⁸ ～10 ⁹ PFU/mL phage content; the diluted composition may contain less active ingredient, for example, 10 ³ ～10 ⁶ PFU/mL phage content; medium content isFor example, it may be 10 ⁶ ～10 ⁸ PFU/mL. In addition, other suitable ingredients, such as the various biologically acceptable carriers listed above, may be included. According to an embodiment of the present application, the phage composition of the present application has a dose of the phage of 10 or more ³ PFU/mL is relatively suitable, although the application is not so limited, as some specific applications are contemplated.

If desired, other active biocides (which may be biological biocides or chemical biocides such as antibiotics) may also be included in the compositions of the present application to achieve co-killing of phage-specific pathogens of the present application as well as other pests in a single use.

The phage of the application, host cells containing it, or compositions containing it, may also be contained in a container or kit. Preferably, the kit further comprises instructions for use, etc., to facilitate application by those skilled in the art.

The phage preparation is in the form of oral solid preparation, liquid preparation or freeze-dried preparation, and can be applied to prevention and/or treatment of aeromonas salmonicida through oral administration, medicated bath, injection and other modes.

The application has the main advantages that:

(1) In the application, the phage capable of efficiently lysing bacterial hosts is screened and separated, and has strong lytic effect on pathogenic bacterial hosts in aquaculture environment.

(2) The phage provided by the application has strong infection capability; the incubation period for aeromonas salmonicida was only 20 minutes with an outbreak of about 284 PFU/cell; the incubation period is short, the cleavage amount is high, and the phage is the preferred phage for inhibiting aeromonas salmonicida. It can maintain good activity in pH=5.0-9.0, and phage titer can be maintained at 10 ⁸ PFU/mL, with the rising or decreasing pH value, the phage potency is significantly reduced, but it has better in acidic or alkaline environmentStability. And has good host specificity, which is beneficial to environmental safety.

(3) The phage provided by the application can be used for industrialized mass production, and can be specifically amplified by host bacterium aeromonas salmonicida; the phage can also be used as a disinfectant for disinfecting water bodies and culture ponds of a farm, thereby treating animal farm pollution; the phage is also useful for the preparation of medicaments for the prevention and treatment of diseases caused by its bacterial host, in particular aeromonas salmonicida.

In conclusion, the phage provided by the application has high-efficiency infection and strong cracking, has good tolerance to acidic and alkaline environments, has good adaptability to high and low temperatures, and has very ideal application prospects in aquaculture.

The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental methods, in which specific conditions are not noted in the following examples, are generally according to conventional conditions such as those described in the guidelines for molecular cloning experiments, or according to the manufacturer's recommendations.

EXAMPLE 1 isolation and identification of Aeromonas salmonicida AS01

In order to obtain organisms which effectively kill fish germs, the inventor conducts research for many years and large-scale screening. The inventor separates a salmonicida aeromonas strain from turbot disease fish in a turbot farm in Liaoning, and the salmonicida aeromonas strain is named AS01. By taking the aeromonas salmonicida AS01 AS a host, the inventor separates the aeromonas salmonicida phage from the environment to obtain a aeromonas salmonicida phage which is named AS vB_ AsM _ZHF.

The inventor samples liver and spleen tissues from diseased fish, performs four-region streak on TSA solid culture medium, performs inversion culture at 28 ℃ for 24-48 hours, picks up bacterial colonies growing on a flat plate for gram staining, picks up bacterial colonies of gram negative short bacillus on the flat plate for streak again after observation under a microscope, repeats the steps for 3 times until bacterial colonies with uniform size and shape are formed on the flat plate, then picks up single bacterial colonies to inoculate in 5mL of TSA culture medium, and performs shaking culture at 28 ℃ for 12 hours at 200rpm to obtain turbid bacterial suspension.

Then, the inventor carries out physiological and biochemical identification on the obtained bacterial liquid, and carries out sequencing and sequence comparison on 16sRNA thereof, and determines that the bacterial strain is aeromonas salmonicida, and the 16sRNA sequence is as follows (SEQ ID NO: 1):

tgcagtcgagcggcagcgggaaagtagcttgctacttttgccggcgagcggcggacgggtgagtaatgcctgggga

tctgcccagtcgagggggataacagttggaaacgactgctaataccgcatacgccctacgggggaaaggaggggac

cttcgggcctttcgcgattggatgaacccaggtgggattagctagttggtggggtaatggctcaccaaggcgacga

tccctagctggtctgagaggatgatcagccacactggaactgagacacggtccagactcctacgggaggcagcagt

ggggaatattgcacaatgggggaaaccctgatgcagccatgccgcgtgtgtgaagaaggccttcgggttgtaaagc

actttcagcgaggaggaaaggttggcgcctaatacgtgtcaactgtgacgttactcgcagaagaagcaccggctaa

ctccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattactgggcgtaaagcgcacgcaggc

ggttggataagttagatgtgaaagccccgggctcaacctgggaattgcatttaaaactgtccagctagagtcttgt

agaggggggtagaattccaggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggcccc

ctggacaaagactgacgctcaggtgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgta

aacgatgtcgatttggaggctgtgtccttgagacgtggcttccggagctaacgcgttaaatcgaccgcctggggag

tacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatg

caacgcgaagaaccttacctggccttgacatgtctggaatcctgcagagatgcgggagtgccttcgggaatcagaa

cacaggtgctgcatggctgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccctgt

cctttgttgccagcacgtaatggtgggaactcaagggagactgccggtgataaaccggaggaaggtggggatgacg

tcaagtcatcatggcccttacggccagggctacacacgtgctacaatggcgcgtacagagggctgcaagctagcga

tagtgagcgaatcccaaaaagcgcgtcgtagtccggatcggagtctgcaactcgactccgtgaagtcggaatcgct

agtaatcgcgaatcagaatgtcgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtg

ggttgcaccagaagtagatagcttaaccttcgggagggcgttac

the present inventors designated this strain AS AS01 and stored it in a refrigerator at-80 ℃.

Example 2 isolation and identification of Aeromonas salmonicida phage vB_ AsM _ZHF

(1) Sample pretreatment

Sampling wastewater in a turbot farm in Liaoning, filtering the collected water sample by using 8 layers of gauze, centrifuging the filtrate at the temperature of 4 ℃ for 5min at 8000 Xg, filtering the centrifuged supernatant by using a microporous filter membrane with the pore diameter of 0.22 mu m, and preserving at the temperature of 4 ℃ for later use.

(2) Enrichment culture of phage samples

40ml of the filtrate, 10ml of 5 XSSC medium and 1ml of aeromonas salmonicida AS01 medium in the logarithmic growth phase were taken, added to 250ml shake flasks and cultured overnight at 100rpm at 28 ℃. 1ml of culture solution is taken, 8000 Xg is centrifugated for 5min, and the supernatant after centrifugation is filtered and sterilized by a microporous filter membrane with the pore diameter of 0.22 mu m.

(3) Phage isolation

The filtrate is subjected to gradient dilution by using SM buffer solution, 0.5mL of the dilution solution and 0.2mL of aeromonas salmonicida AS01 bacterial suspension are taken to be mixed and incubated for 10min at normal temperature, then semi-solid TSB culture medium with the temperature of about 55 ℃ is added, and after uniform mixing, the mixture is poured onto a TSA solid plate which is prepared in advance. Culturing at 28deg.C for 12-36 hr. The presence of plaque on the bilayer plate was observed, and if plaque was present, the presence of phage was indicated.

(4) Phage spot purification

Plaques were picked with a sterile gun head and repeatedly blown in 1mL SM buffer, incubated overnight at 4 ℃, then phage extracts were gradient diluted and observed for plaque morphology by double-layer plate method. The above purification steps were repeated three times to obtain a phage of the same size in phage form, which was designated vB_ AsM _ZHF. The plaque schematic of vB_ AsM _ZHF is shown in FIG. 1. As can be seen from FIG. 1, the salmonicida aeromonas phage vB_ AsM _ZHF forms a plaque with a transparent circular shape and a halo around the plaque, and the diameter of the plaque is about 3.0-3.5 mm.

(5) Filtering, sterilizing and preserving

Centrifuging the purified phage culture solution at 8000 Xg for 5min, filtering and sterilizing the supernatant with microporous membrane with pore diameter of 0.22 μm, adding glycerol to the filtrate to obtain final concentration of 30%, and storing at-80deg.C.

(6) Determination of phage titers

Centrifuging 8000 Xg of phage culture solution for 5min, filtering the supernatant with 0.22 μm filter membrane, subjecting the filtrate to gradient dilution with SM buffer solution, preparing double-layer plate with Aeromonas salmonicida AS01 AS host bacteria, and culturing at 28deg.C under inverted condition.

Phage titer (pfu/mL) =number of plaques x dilution.

The potency of the phage can reach 10 by measurement ⁹ PFU/mL。

Example 3 morphological observations of phages

Phage samples were pre-treated with phosphotungstic acid negative staining. 20 mu L of phage culture liquid is dripped on a copper mesh after being filtered by a 0.22 mu m filter membrane, and the redundant liquid is sucked by a piece of absorbent paper after 10 min. After standing at room temperature for 2min, 20. Mu.L of 2% phosphotungstic acid dye was added dropwise to the copper mesh, and immediately after dyeing for 30s, the excess phosphotungstic acid solution was sucked off with a piece of absorbent paper. The phage morphology was observed by transmission electron microscopy at room temperature for 5 min.

An electron micrograph of the Aeromonas salmonicida phage vB_ AsM _ZHF is shown in FIG. 2. According to FIG. 2, the head of phage vB_ AsM _ZHF is an icosahedron, and the tail consists of a hollow structure and an outer sheath. The phage had a head length of 95nm, a width of 91nm and a tail length of 109nm.

Analysis was based on the rules of phage isolation and naming, and the morphology of the phage, which belongs to the family of myotail phages, the order of the end phages.

Example 4 optimal multiplicity of infection determination

According to 1: 100. The aeromonas salmonicida AS01 bacterial suspension in logarithmic growth phase was added to TSB medium at a multiplicity of infection of 1: 1. 1:10. 1: 100. 10: 1. 100: 1. 1000:1, adding phage culture solution into TSB culture medium, shake culturing at 28deg.C for 8 hr at 100rpm, centrifuging at 4deg.C for 5min at 8000 Xg, filtering and sterilizing the supernatant with microporous membrane with pore size of 0.22 μm, and measuring titer by double-layer plate method.

The results of the complex infection experiments with the aeromonas salmonicida phage vb_ AsM _zhf are shown in figure 3. According to FIG. 3, phage titers were highest when the multiplicity of infection was 1:10 (i.e., MOI of 0.1 in the figure). The measurement result shows that the infection complex number of vB_ AsM _ZHF can be 100:1-1:1000; relatively preferred multiplicity of infection is 1:1 to 1:100; wherein the optimal multiplicity of infection is 1:10.

Example 5 determination of phage growth curve

Phage were mixed with 1ml of aeromonas salmonicida suspension at a multiplicity of infection of 1:100, incubated for 10 minutes, and centrifuged at 12,000Xg for 2 minutes at 4 ℃. The pellet was resuspended with 1ml of TSB medium and repeated 3 times. The suspension was added to 10ml TSB and incubated with shaking at 28 ℃. Phage titers were determined by the double-layer plate method, with phage titers being determined every 10 minutes. The burst size was calculated by dividing the final phage titer by the initial phage titer.

The one-step growth profile of the aeromonas salmonicida phage vb_ AsM _zhf is shown in fig. 4. According to FIG. 4, there was no significant change in the titer of the phage within 20min after infection with the host bacteria, indicating a incubation period of about 20min, and the titer of the phage gradually increased within 20-60min after infection, indicating a phage lysis period of about 40min.

Through calculation, the splitting amount of the phage is 284 PFU/cell, which shows that the phage has strong replication capacity and splitting capacity.

Example 6 acid-base resistance experiment

Adjusting pH of physiological saline to 3, 4, 5, 6, 7, 8, 9, 10, 11 with dilute HCl solution and dilute NaOH solution, and adjusting pH to 10 with the above solutions with different pH values ¹⁰ PFU/mL phagesDiluting the bulk solution to 10 ⁸ PFU/mL, the dilutions were incubated at 4℃for 2 hours, and the phage titers were determined by double-plate assay after gradient dilution with SM buffer solution.

The results of the acid-base resistance experiment of the aeromonas salmonicida phage vB_ AsM _ZHF are shown in FIG. 5. As can be seen from fig. 5, in systems with different pH values, phage vb_ AsM _zhf can maintain good activity in the ph=5.0-8.0 range. With the rise and fall of the pH value, the titer of the phage is reduced to a certain extent. However, at ph=11 and ph=3, the phage still had some activity.

The results demonstrate that the phage vB_ AsM _ZHF of the application has good tolerance to both acidic and alkaline environments.

Example 7 thermal stability experiment

The present inventors will 10 ⁸ PFU/mL phage solutions were incubated at 36℃at 50℃at 60℃at 70℃for 2 hours, respectively, and phage titers were determined by double-layer plate method after gradient dilution with SM buffer solution.

The results of the aeromonas salmonicida phage vb_ AsM _zhf heat stability experiment are shown in fig. 6. From this result, it was found that phage remained stable at 36℃and the phage activity was reduced by about half at 60℃and was completely lost at 70 ℃.

Therefore, the phage vB_ AsM _ZHF of the application can maintain good activity in low temperature (such as 0-10 ℃ C. Or lower) and normal temperature environment (such as 10-28 ℃ C.) and has ideal high temperature tolerance (such as 30-50 ℃ C.).

Example 8 cleavage efficiency experiment

1mL of phage vB_ AsM _ZHF and 1mL of host bacterium AS01 bacterial liquid are taken to be incubated for 10 minutes at 28 ℃, the bacterial liquid is diluted in a gradient mode by SM buffer solution, the diluted liquid is coated on a flat plate to perform flat plate colony counting, and the culture is carried out at 28 ℃ for 24 hours.

Meanwhile, 1mL of SM buffer and 1mL of host bacterium AS01 bacteria were incubated at 28℃for 10 minutes AS a control group.

Phage lysis rate= (1-number of colonies of experimental group/number of colonies of control group) ×100%.

The determination result shows that the cracking rate of the aeromonas salmonicida phage vB_ AsM _ZHF reaches 98%, has a good cracking effect on hosts, and is suitable for being used in the aquaculture process.

Example 9 phage vs host lysis curve

mu.L of bacterial host suspension and phage solution in logarithmic growth phase were added to 100. Mu.L of 2 XTSB medium at a multiplicity of infection of 1:10 in 96 well plates and the final volume was made up to 200. Mu.L with deionized water and cultured with shaking at 28℃for 60h. As a control, no phage solution was added. OD600 was measured with a microplate reader every 1 minute and the results are shown in fig. 7.

According to FIG. 7, when no phage is added to the system, the OD is increased as the host bacteria grow ₆₀₀ The increase was slow in 1 hour, exponential in 1-7 hours, and slow in 7-45 hours, as was the OD600 of the host bacteria when the phage was added to the host, and also increased in the first 1 hour, as the host bacteria entered the logarithmic phase of growth, the phage gradually began to lyse the host, and within 45 hours there was no significant increase in the OD600 of the host bacteria, but then the host bacteria that were not lysed began to grow gradually, and the OD600 of the host bacteria gradually increased.

EXAMPLE 10 evaluation of the Effect of phage treatment on Aeromonas salmonicida infection

The inventors selected turbot (30 g±2 g) AS the test fish, randomly divided into 5 groups, namely 3 Phage treatment groups (as+phase), 1 challenge control group (as+pbs) and 1 blank control group (pbs+pbs), each group having 20 tails. At 8×10 ⁴ The phage-treated group and the challenge control group were subjected to intramuscular injection and challenge by CFU/tail aeromonas salmonicida, and the blank control group was subjected to intramuscular injection of PBS solution at a volume of 100 μl/tail. At the same time at 8×10 ² PFU/tail, 8×10 ⁴ PFU/tail, 8×10 ⁶ The PFU/tail aeromonas salmonicida phage solution was administered by intraperitoneal injection to phage treatment groups, and the challenge control group and the blank control group were injected intraperitoneally with PBS solution at a volume of 100. Mu.L/tail. And (5) observing the death condition of turbot after 20 days of cultivation.

As can be seen from fig. 8, the blank control group had no fish death within 20 days, the poison attack control group had a poison attack period of 6-8 days, and the survival rate was 10%. Phage treatment group at 8The fish gradually die within 16 days, and the administration dose is 8 multiplied by 10 ² The survival rate of the phage-treated group of PFU/tail was 60% and the dose was 8X 10 ⁴ The survival rate of the phage-treated group of PFU/tail was 70% and the dose was 8X 10 ² The survival rate of the phage-treated group of PFU/tail was 75%.

The survival rate of the phage treatment group is obviously higher than that of the virus attack control group, which indicates that the aeromonas salmonicida phage can protect fish bodies from the aeromonas salmonicida.

Meanwhile, the phage is applied, and no toxic or side effect on other organisms in the environment is observed.

Preservation of biological materials

The aeromonas salmonicida phage vb_ AsM _zhf (Aeromonas salmonicida phage vB _ AsM _zhf) of the present application has been deposited at the chinese collection of typical cultures (university of martial arts, china), date of deposit: 2021, 5 and 31 days, and the preservation number is CCTCC NO: M2021631. Viable phages were detected by the collection center.

The foregoing is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and alterations of this application will occur to those skilled in the art. Any modifications, equivalent substitutions, improvements, etc. which are within the spirit and principles of the present application are intended to be included within the scope of the present application.

Sequence listing

<110> university of Industy of Huadong

Biological technology Co.Ltd

<120> isolated Aeromonas salmonicida phage, compositions and uses thereof

<130> 214672

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1412

<212> DNA

<213> Aeromonas salmonicida (Aeromonas salmonicida)

<400> 1

tgcagtcgag cggcagcggg aaagtagctt gctacttttg ccggcgagcg gcggacgggt 60

gagtaatgcc tggggatctg cccagtcgag ggggataaca gttggaaacg actgctaata 120

ccgcatacgc cctacggggg aaaggagggg accttcgggc ctttcgcgat tggatgaacc 180

caggtgggat tagctagttg gtggggtaat ggctcaccaa ggcgacgatc cctagctggt 240

ctgagaggat gatcagccac actggaactg agacacggtc cagactccta cgggaggcag 300

cagtggggaa tattgcacaa tgggggaaac cctgatgcag ccatgccgcg tgtgtgaaga 360

aggccttcgg gttgtaaagc actttcagcg aggaggaaag gttggcgcct aatacgtgtc 420

aactgtgacg ttactcgcag aagaagcacc ggctaactcc gtgccagcag ccgcggtaat 480

acggagggtg caagcgttaa tcggaattac tgggcgtaaa gcgcacgcag gcggttggat 540

aagttagatg tgaaagcccc gggctcaacc tgggaattgc atttaaaact gtccagctag 600

agtcttgtag aggggggtag aattccaggt gtagcggtga aatgcgtaga gatctggagg 660

aataccggtg gcgaaggcgg ccccctggac aaagactgac gctcaggtgc gaaagcgtgg 720

ggagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgtcg atttggaggc 780

tgtgtccttg agacgtggct tccggagcta acgcgttaaa tcgaccgcct ggggagtacg 840

gccgcaaggt taaaactcaa atgaattgac gggggcccgc acaagcggtg gagcatgtgg 900

tttaattcga tgcaacgcga agaaccttac ctggccttga catgtctgga atcctgcaga 960

gatgcgggag tgccttcggg aatcagaaca caggtgctgc atggctgtcg tcagctcgtg 1020

tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ctgtcctttg ttgccagcac 1080

gtaatggtgg gaactcaagg gagactgccg gtgataaacc ggaggaaggt ggggatgacg 1140

tcaagtcatc atggccctta cggccagggc tacacacgtg ctacaatggc gcgtacagag 1200

ggctgcaagc tagcgatagt gagcgaatcc caaaaagcgc gtcgtagtcc ggatcggagt 1260

ctgcaactcg actccgtgaa gtcggaatcg ctagtaatcg cgaatcagaa tgtcgcggtg 1320

aatacgttcc cgggccttgt acacaccgcc cgtcacacca tgggagtggg ttgcaccaga 1380

agtagatagc ttaaccttcg ggagggcgtt ac 1412

Claims

1. An isolated phage having a collection number of CCTCC No. M2021631 in the chinese collection of typical cultures.

2. Use of a bacteriophage of claim 1 for the preparation of a composition for inhibiting a bacterial host thereof; the bacterial host is aeromonas salmonicida.

3. The use of a bacteriophage of claim 2, wherein said composition is a pharmaceutical composition, a detergent and/or a disinfectant.

4. The use of a bacteriophage of claim 3, wherein said pharmaceutical composition is a vaccine composition that prevents fish diseases associated with bacterial host infection of said bacteriophage, including scabies caused by aeromonas salmonicida infection.

5. The use of a bacteriophage of claim 3, wherein said pharmaceutical composition is a therapeutic composition for treating a fish disease associated with bacterial host infection of said bacteriophage, said fish disease comprising: scabies caused by aeromonas salmonicida infection.

6. The use of a bacteriophage of claim 3, wherein the cleaning or sanitizing agent is an environmental cleaning or sanitizing agent.

7. A composition for inhibiting aeromonas salmonicida comprising the isolated phage of claim 1.

8. The composition of claim 7, further comprising a biologically acceptable carrier.

9. The composition of claim 8, wherein phage in the composition is greater than or equal to 10 ³ PFU/mL。

10. The composition of claim 9, wherein the phage in the composition is 10 ³ ～10 ¹⁰ PFU/mL。

11. The composition of claim 10, wherein the phage in the composition is 10 ⁴ ～10 ⁹ PFU/mL。

12. The composition of claim 7, wherein the composition is a pharmaceutical composition, a cleaning agent, or a disinfectant.

13. The composition of claim 12, wherein the cleaning or sanitizing agent is an environmental cleaning or sanitizing agent.

14. The composition of claim 12, wherein the formulation of the composition comprises: liquid or solid formulations.

15. The composition of claim 12, wherein the formulation of the composition comprises: freeze-dried agent, aqueous solution, emulsion, suspending agent, powder, granule or microcapsule.

16. The composition of claim 15, wherein the aqueous solution is a sprayable solution.

17. The composition of claim 15, wherein the powder is a wettable powder.

18. The composition of claim 12, wherein the pH of the composition is from pH3.0 to 11.0.

19. The composition of claim 18, wherein the pH of the composition is from pH4.0 to 9.0.

20. A method of using the phage preparation composition of claim 1, comprising: amplifying and culturing the phage of claim 1, and mixing the phage with a carrier, wherein the carrier is a biologically acceptable carrier.

21. The method of claim 20, wherein the amplification culture comprises: the phage is inoculated with its bacterial host culture so that the phage infects its host and replicates, and its bacterial host is aeromonas salmonicida.

22. A method of non-therapeutically inhibiting a pathogenic microorganism, the method comprising: treating a subject in need of bacterial inhibition with the phage of claim 1; the object is a region or an appliance to which bacteria are attached, and the region is public places or fish culture water; the bacteria are aeromonas salmonicida which can be infected by the phage and then lysed; the subject is not a human or animal.

23. The method of claim 22, wherein the treatment is performed at a ph of 3.0 to 11.0.

24. The method of claim 23, wherein the treatment is performed at a pH of 4.0 to 9.0.

25. The method of claim 22, wherein the treatment is performed at a temperature of 0 to 50 ℃.

26. The method of claim 25, wherein the treatment is performed at a temperature of 0 to 40 ℃.

27. The method of claim 26, wherein the treatment is performed at a temperature of 0 to 30 ℃.

28. A kit or kit for inhibiting aeromonas salmonicida, said kit or kit comprising:

the phage of claim 1; or (b)

The composition of any one of claims 7 to 19.